Gbeta5 is required for normal light responses and morphology of retinal ON-bipolar cells

Abstract

Gbeta5 exists as two splice variants, Gbeta5-S and Gbeta5-L, which interact with and stabilize the R7 members of the regulators of G-protein signaling (RGSs): RGS6, RGS7, RGS9, and RGS11. Although the role of Gbeta5-L and RGS9-1 is established in photoreceptors, the physiological functions of Gbeta5-S and other R7 RGS proteins remain unclear. We found that the electroretinogram of Gbeta5-/- mice lacks the b-wave component and that Gbeta5-S and RGS11 colocalize with Go alpha at the tips of the ON-bipolar cell dendrites. Unexpectedly, we found a significant reduction in the number of synaptic triads in the outer plexiform layer (OPL) of the Gbeta5-/- mice, which is evident at postnatal day 14. Transgenic expression of Gbeta5-L in rods failed to rescue the b-wave or the OPL defects. These results indicate that Gbeta5-S is indispensable for OPL integrity and normal light responses of the retina.

Figure 1.

The postphotoreceptoral electroretinographic no-b-wave phenotype of the Gβ5^−/− mice. A, Representative scotopic ERG responses of dark-adapted adult control (left) and Gβ5^−/− mice (right) to a series of white flashes with indicated intensities in log cd s/m². B, DNA construct used for the generation of TGβ5L transgenic mice. C, Western blots showing Gβ5 and RGS9-1 levels in 10 μg extracts of control, Gβ5^−/−, and TGβ5L^−/− mouse retinas. D, Image of a TGβ5L^−/− retinal section probed with a Flag antibody showing outer segment localization of the transgenic product. OS, Outer segment; IS, inner segment. Scale bar, 10 μm. E, Representative scotopic ERG responses of dark-adapted control (left) and TGβ5L^−/− mice (right), treated or untreated with APB.

Figure 2.

Localization of Gβ5, RGS7, and RGS11 in the retina and abnormal ON-bipolar cell morphology of the Gβ5^−/− mice. A–D, Retinal sections derived from control (A–C) and Gβ5^−/− (D) mice probed for Gβ5 (A, D), RGS7 (B), and RGS11 (C). Sources and dilutions of antibodies are shown in Table 1. Scale bar, 10 μm. E, F, Colocalization of Goα with Gβ5 (E) and RGS11 (F) in control retinas. Scale bar, 5 μm. G, H, Retinal sections from age-matched adult control (G) and Gβ5^−/− animals (H) colabeled with mGluR6 and PKCα. Scale bar, 10 μm.

Figure 3.

Abnormal OPL ultrastructure of the Gβ5^−/− mice. A, Western blot analyses of 10 μg retinal extracts of control mice at indicated postnatal ages showing relative levels of Gβ5-S and Gβ5-L in reference to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level. B, TEM images of the OPL of the control (top) and Gβ5^−/− (bottom) mice. Scale bar, 5 μm. RS, Rod spherule; CP, cone pedicle. Asterisk denotes abnormal structures found in the Gβ5^−/− retina. Representative OPL synapses are shown in insets. C, D, The averaged percentage of rod spherules with normal synaptic triads (C) and ribbons (D) at indicated postnatal days. Error bars are SEM (n = 3). E, The averaged number of synaptic triads and ribbons found per cone pedicle per section in adult control and Gβ5^−/− mice, n = 3. F, The averaged percentage of rod spherules with triads and ribbons in adult TGβ5L^−/− mice compared with age-matched Gβ5^−/− and control animals, n = 3.